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Genetic Analysis of the Shaker Gene Complex of Drosophila Melanogaster

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Genetic Analysis of the Shaker Gene Complex of Drosophila Melanogaster Copyright 0 1990 by the Genetics Society of America Genetic Analysisof the Shaker Gene Complexof Drosophila melanogaster A. Ferriis,* S. LLamazares,* J. L. de la Pornpa,* M. A. Tanouye? and0. Pongs* *Institute Cajal, CSIC, 28006 Madrid, Spain, +California Instituteof Technology, Pasadena, California 91 125, and 'Ruhr Universitat,Bochum, Federal Republic of Germany Manuscript received May 10, 1989 Accepted for publication March 3, 1990 ABSTRACT The Shaker complex (ShC) spans over 350 kb in the 16F region of the X chromosome. It can be dissected by means of aneuploids into three main sections: the maternal effect (ME), the viable (V) and the haplolethal (HL) regions. The mutational analysis of ShC shows a high density of antimorphic mutations among 12 lethal complementation groupsin addition to 14 viable alleles. The complex is the structural locus of a family of potassium channels as well as a number of functions relevant to the biology of the nervous system. The constituents of ShC seem to be linked by functional relationships in view of the similarity of the phenotypes, antimorphic nature of their mutations and the behavior in transheterozygotes. We discuss the relationship between the genetic organization of ShC and the functional coupling of potassium currents with the other functions encodedin the complex. HAKER was the first behavioral mutant detected do not appear tohave the capability of generating, by S in Drosophila melanogaster (CATSCH1944). It was themselves, gated ionic channels. named after another mutantwith a similar phenotype K+ currents are known to be themost diverse class isolated earlier in Drosophila funebris (LUERS 1936). of ionic currents in terms of kinetics, pharmacology The original phenotype was described as the trem- and sensitivity (HILLE 1984; RUDY 1988).Also, these bling of appendagesin the anesthetized fly. The elec- currents are known to play an essential role in many trophysiological study of most viable Sh alleles has aspects of the biology of organisms frommorpho- revealed a number of functional defects including: genesis (JAFFE 1979; KLINE,ROBINSON and NUCCI- excess of neurotransmitter release at the neuromus- TELLI 1983) tomodulation of synaptic efficacy during cular junction (JAN,JAN and DENNIS1977), abnormal learning(LEVITAN 1988; KANDELand SCHWARTZ actionpotentials in the cervical giantfiber (CGF) 1982).In addition and in contrast toother ionic (TANOUYE,FERR~S and FUJITA 1981; TANOUYEand currents, K+ currentsare ubiquitous amongorga- FERR~S1985) and absence or altered kinetics of the nisms, tissues and developmental stages. fast voltage dependent transient K+ current (Ia) in Shaker was known to be a gene complex from the muscles under voltage clamp conditions(SALKOFF and beginning of its genetic analysis (TANOUYE,FERR~S WYMAN 1981; Wu and HAUGLAND 1985)well as as in and FUJITA198 1). We haveentertained thehypothesis single channel studies of dissociated nervous systems that thediversity of K+ currents has its counterpart in (SOLC,ZAGOTTA and ALDRICH1987). The subsequent the complexity of the Shaker locus. Now that one of molecular analysis of this locus has shown the exist- the originalpropositions has beendemonstrated, ence of a complex transcription unit from which a namely that ShC is the structural locus for a number large family of products is generated by meansof of K+ channels, it seems appropriate to dissect ShC differential and/oralternative splicing mechanisms into its genetic components, to study the biology of (BAUMANNet al. 1987; TEMPELet al. 1987; KAMB, their mutations and tobegin to unravel the functional IVERSON andTANOUYE 1987). Most of these products relationships among thesecomponents. Thereare have structural features compatible with membrane abundant examples in a variety of preparations illus- proteins (PONGSet al. 1988; SCHWARTZet al. 1988) trating how K+ currents are modulated by means of and some of them have beendemonstrated tofunction phosphorylation,neuropeptides, cyclic nucleotides, as K+ channels afterRNA expression in Xenopus etc. (LOGOTHETISet al. 1987; NORTH et al. 1987; oocytes (TIMPEet al. 1988; IVERSONet al. 1988). ASHCROFT1988). Synaptic efficacy is largely based on Different RNAs give rise to K+ currents of different the diversified modulationof K+ channels (CROW characteristics depending on the specific 3' and 5' 1988; LEVITAN1988). In this context wewill ask if exon combination present (KAMB, TSENG-CRANKand the requiredfunctional coupling among these diverse TANOUYE1988). Thus, it seems that this transcription activities is related to thegenetic organization ofShC. unit from Shaker encodes a variety of K+ channels as We do not define Shaker solely on the basis of the well as a number of putative membrane proteins that K+ currents phenotype. Also, we do not assume that Genetics 125: 383-398 (June, 1990) 384 Ferriis et al. the Sh products participate exclusively in the forma- tizedflies were observed for 30 min or longer during tion of ion channels. We find untenable such a one- recovery under the dissectin microsco e The following Sh to-onecorrespondence between geneproducts and viable alleleswere tested: ShF , Sh'", Sh"';' and ShrK"'*O. Electrophysiology: Action potentials were registered biological features. Rather, we define a genecomplex from the CGF interneuron (KOTO et al. 1981). The proce- in broader terms as the portion of the DNA where dure for dissection, experimental conditions and character- clustered mutations show genetic relationships andl isticsof the preparation canbe found in TANOUYEand or similar phenotypes at any given level of observa- FERR~S(1 985). tion. Mosaics: Gynandromorphs were obtained from the prog- eny ofcand/cand females or among R(I)Z,ln(I)w""/*embryos. The resulting mosaics were routinely analyzed for general MATERIALS AND METHODS behavior including possible shaking activity. Also,the extent of maleffemale territories were drawn on standard fly Mutants, rearrangements, mutagenesis and nomencla- sketches. Each gynandromorph was either mounted in Eu- ture: The description of mutants and rearrangements used para1 for detailed cuticular observation or processed for in this study can be found in LINDSLEYand GRELL(1968), histology. A tentative fate map of the mutant focus of each LINDSLEYand ZIMM (1985, 1986, 1987) or TANOUYE,FER- lethal was calculated with the limited collection of gynan- Rirs and FUJITA(1981). Also, Table 1 and Figure 1 show a dromorphs as described in HOTTAand BENZER(1 972). summary of the rearrangements used. Somatic recombination cloneswere induced by X-ray The following agents were used as mutagens under the (Philips MG 151 Be, 150 r/min, 100 kV, 15 mA and 2 mm conditions of LEWISand BACHER(1 968) or AUERBACHand AI filter at a total dose of 1000 r) in heterozygous larvae of MOSER (1953): ethyl methanesulphonate (EMS) (250 mM), the indicated age. Germ line clones were induced by the ethyl nitrosourea (ENU) (250 mM), formaldehyde (F), die- same procedure in FS(I)KS1237 v/* larvae irradiated 24- poxy butane (DEB) (50 mM) and X-ray (4000 r). Unless 48 h after egg laying. The use of this agametic dominant otherwise indicated, 4-7 days oldf' os males were treated. female sterile mutant (FS(I)KS1237)allows the detection of The T(X;Y)'s wereinduced by J. Merriam on ay+P.YLBs a germ line clone bearing female as a fly that under COP chromosome. The proximal and distal elements canbe anesthesia relaxes the genital muscles and liberates a re- obtained separatedly and are designated by superscript P or tained egg. Under these conditions 6% of the irradiated D. Males of the constitution X/V7'/Y are sterile due to the control females carry a germ line clone (WIESCHAUS1980). hiperploidy of the base of the X. We mutagenized YsX.YL The development of each egg laid by these mosaic females compounds with X-ray in order to obtain deficiencies of the was analyzed individually. base of the X chromosome as fertile males YsX YL*/V7'. In Lethal phase and whole mounts: Fecund females were this way Dj(I)S4010was obtained. In(I)Px (Panoramix)was allowed to lay eggs for 20-hr periods at 25" and 80% obtained from the progeny of X-ray treated os males and humidity on regular fly food plates containing 5% sucrose detected by thedominant, larger than normal, eyesize and a drop of live yeast. Eggs were collected, counted and phenotype. transferred to petri dishes containing filter paper soaked in The mutations located within ShC are named after the 5% sucrose and a few drops of yeast. Groups of 20-30 eggs region to which they map: ME, V or HL for maternal effect, were examined at 1-day intervals; thus, the fraction of dead viable and haplo lethal respectively, followedby the number individuals was estimated in 24-hr periods. Routinely, the of complementation group and the code number. For in- lethal embryos were cleared and mounted for observation stance, ShMI.".3u5indicates a Shaker mutation of the first group (VAN DER MEER, 1977). The classificationof lethals as of complementation (from distal to proximal) in the ME recessive, semidominant or dominant is according to region, isolated as code number 305. HADORN(1 955). Characterization of the shaking activity. The intensity of appendage vibration in the various genotypes listed in RESULTS Table 5 was rated1, 2 or 3 in increasing order. These estimations are based in the shaking activity of the meso- thoracic legs of groups of ether-anesthetized (30 sec) flies. Aneuploid analysis:In the process of analyzing the Care was taken to use flies not older than five days and not ShCwe have used a number of chromosomalre- younger than one day. Aged Sh mutant adults (over 10 days) arrangements (see Figure 1) with breakpoints in the exhibit progressively more intense, although erratic, appen- vicinity of the Shaker locus. In order to identify their dage vibration, possibly related to the conspicuous impair- linear order and the existence of lack-of-function le- ment of muscle structure observed in several Sh alleles. Also, very young Sh adults (1 day) do not express the shaking thals, we constructed aneuploids in all feasible com- phenotype in full magnitude.
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